Project Summary This proposal has three specific aims that address key unsolved problems in thiamin metabolism. Aim 1 will continue our exploration of the remarkable mechanistic enzymology of thiamin pyrimidine formation. This reaction, catalyzed by ThiC in bacteria and by THI5 in Yeast, is the most complex unresolved rearrangement reaction in primary metabolism. Our approach will center on using substrate analogs to trap intermediates for chemical and EPR characterization. Improved understanding of the thiamin pyrimidine biosynthesis is important for fundamental reasons as well as the development of strains for the production of thiamin by fermentation. In Aim 2, we will turn our attention to the relatively understudied area of cofactor catabolism and elucidate the enzymology of thiamin catabolism. The knowledge acquired from these catabolic studies will be used to evaluate the importance of thiamin catabolism in the gut microbiome and its relation to thiamin deficiency disease. In the third research aim, we will leverage our understanding of thiamin synthesis and metabolism to design thiamin analogs that can be used for PET imaging and for selective labeling of thiamin-utilizing enzymes in a proteome. These studies will allow us to explore the use of thiamin as an imaging agent and may prove useful in diagnostics.